(I) Equine Infectious Anemia
Equine infectious anemia (hereinafter "EIA") is a disease that primarily affects horses and ponies and is commonly known as swamp fever. EIA or swamp fever is a progressive disease of horses and other Equidae. The disease is caused by a virus in the lentivirus subfamily of the retrovirus family. The EIA virus infection results in a chronic disease whose main pathology is the immune-mediated destruction of red blood cells. The persistent disease can progress through acute, subacute, chronic and inapparent forms. The virus persists and escapes destruction by the host animal by hiding in macrophages and by mutating into variant strains. Diagnosis is the major means for the prevention and control of the disease because there is no vaccine or cure for EIA. The disease has been controlled in the United States by a federally regulated test and isolation/slaughter program. The official test for this federally regulated program is the Coggins agar gel immunodiffusion test (hereinafter "AGID test") (see: U.S. Pat. Nos. 3,929,982 and 3,932,601). The object of this test is to detect antibody against the EIA virus because antibodies against the virus can be detected more easily over a longer period of time than can the virus itself. The EIA virus antigen forms a visible precipitin line with antibodies specific for EIA virus. However, U.S. Pat. Nos. 3,929,982 and 3,932,601 do not identify the specific virus antigens that are involved in the test.
The EIA virus contains four major non-glycosylated-proteins designated as p26, p15, p11 and p9, and two glycoproteins designated as gp90 and gp45 see: Montelaro, C. J., et al, J. Virol. 42:1029-1038 (1982)). p26 is known as the core antigen and is the predominant protein found in the core of EIA virus. The Coggins AGID test detects mostly p26-specific antibody. The Coggins AGID test is effective as a diagnostic tool because p26 does not mutate and antibody to p26 persists in the blood of horses throughout the course of the disease and for up to seven years. In contrast, systems which directly detect virus antigen have not been developed because of the transient viremia (see: Issel, C. J., et al, J. Am. Vet. Med. Assoc., 1974:727 (1979)).
A problem in the Coggins AGID test is the appearance of nonspecific precipitin lines that are formed by antibodies against contaminating antigens (i.e. non-EIA antigens) in the manufacture of virus preparations. Recently, this problem has been solved by using novel techniques to prepare purified EIA virus antigen (see: U.S. patent application Ser. No. 607,895, filed May 7, 1984). This new version of the AGID test has been licensed and is currently manufactured by TechAmerica Diagnostics under the DiaSystems-EIA label. However, this new AGID test and the Coggins AGID test are disadvantageous for the following reasons: (1) The formation of clearly visible precipitin lines takes 24-48 hours; (2) The performance of the AGID test is labor intensive requiring the laboratory technician to make agar plates, prepare seven-hole patterns on the plates, and perform laborious visual interpretations of the results; (3) The AGID tests detect virus-antigen-specific antibody and cannot directly detect virus antigen. As a result, the early viremia associated with this disease cannot be detected by these AGID tests even though the horse may show early EIA symptoms with no antibody response; and (4) The sensitivity of these AGID tests is limited to antibody concentrations which either form precipitin lines or cause bends in the reference serum lines.
(II) Acquired Immune Deficiency Syndrome
The human T-cell leukemia viruses (hereinafter "HTLV") are structurally similar to the EIA virus and contain a single stranded RNA genome and a RNA-dependent DNA polymerase (reverse transcriptase). Both HTLV and the EIA virus are part of the family retroviridae and are commonly referred to as retroviruses. The HTLV-III virus has been isolated and causally linked to AIDS (see: Popovic, M., et al, Science 224:497-500 (1984); Gallo, R. C., et al, Science 224:500-503 (1984); Schupbach, J., et al, Science 224:503-505 (1984); Sarngadharan, M. G., et al, Science 224:506-508 (1984)) and appears to be identical to the lymphadenopathy-associated virus (hereinafter "LAV") which has also been linked to AIDS (see: Montagnier, L., et al, In Human T-Cell Leukemia/Lymphoma Virus, Gallo, R. C., Essex, M., and Gross, L., eds., Cold Spring Harbor, N.Y.; Cold Spring Harbor Laboratory), pp. 363-370(1984)). The morphology of HTLV-III resembles EIA virus and visna-virus (both lentiviruses) more than HTLV-I or HTLV-II (see: Gonda, M. A., et al, Science 227:173-177 (1985)). HTLV-III has at least three glycosylated proteins designated as gp160, gp120 and gp41 (gp46) and three nonglycosylated proteins designated as p70, p55 and p24 (Robey, W. G., et al, Science 228:593-595 (1985)). p24 is known as the core antigen and is the major structural protein found in the mature virion core of HTLV-III. p24 of HTLV-III corresponds to p26 of EIA, the major structural protein of the EIA virus core.
The similarity of LAV and EIA virus morphology has been independently observed (see: Brun-Vezinet, F., et al, Science 226:453-456 (1984)). In addition, sera from EIA virus-infected horses precipitate the p24 core protein of LAV (see: Montagnier, L., et al, Science 225:63-66 (1984) and Montagnier, L. et al, In Human T-Cell Leukemia/Lymphoma Virus, Gallo, R. C., Essex, M., and Gross, L., eds. (Cold Spring Harbor, N.Y.; Cold Spring Harbor Laboratory), pp. 363-370 (1984)). Thus, it has been demonstrated that antigenic cross-reactivity between EIA virus and HTLV-III exists (see: Casey, J. M., et al, J. Virol. 55:417-423 (1985)). Hence, morphological and antigenic similarities exist between HTVL-III (LAV) and EIA virus. Therefore, CELISA methods developed for EIA antibody and antigen detection are believed to be effective for AIDS antibody and antigen detection.
(III) Other Retroviruses
As discussed above, retroviruses include the subfamily lentivirus. Specific lentiviruses include progressive pneumonia virus (see: Kennedy, R. C., et al, Virology. 35:483 (1968)), and caprine arteritis and encephalitis virus. There is limited immunological cross-reactivity between EIA virus and these viruses (see: Brahic, M., et al in Comparative Diagnosis of Viral Diseases, Vol. 14, Kurstak, E., and Kurstak, C., eds., pp. 619-643, Academic Press, New York, (1981)). It is believed that this cross-reactivity involves the core antigen of these viruses. Thus, CELISA of the present invention directed to measuring the core antigen of EIA and antibodies specific thereto is also believed to be applicable to other retroviruses. In any event, the core antigen of these other retroviruses can also be purified and employed in the present invention to measure the presence thereof in test samples as disclosed in more detail below.
(IV) Antigen Conjugation Systems
Enzyme-labeled antigen has been used for detection of virus-specific antibody. These conjugates are used in antibody capture assays in which an antibody is captured on a solid matrix by an antibody-specific antibody and then subsequently detected by the conjugated antigen (see: U.S. Pat. Nos. 4,273,756 and 4,347,311. This method has been utilized for detecting virus-specific antibody to rubella virus (see: Bonfanti, C., et al, J. Clin. Micro. 21:963-968 (1985); cytomegalovirus (see: Schmitz, H. et al. J. Gen. Virol. 50:59-68 and Van Loon, A. M., et al, J. Clin. Micro. 13:416-422 (1981); Epstein-Barr virus (see: Schmitz, H., J. Clin. Micro. 16:361-366 (1982); varicella-zoster virus (see: Sundqvist, V., J. Virol. Methods 5:219-227 (1982); and flavivirus (see: Schmitz, H., et al, J. Clin. Micro. 19:664-667 (1984)). This method has also been used to detect class-specific and virus-specific antibody to hepatitis virus (see: U.S. Pat. No. 4,273,756).
One disadvantage of the above-described systems is that they cannot detect antigen since they rely solely on antibody capture. A second disadvantage of those systems is that detection of antibody against a single epitope (i.e., antigenic determinant) is difficult because the systems rely upon purified antigen for specificity rather than a monoclonal antibody. A third disadvantage of those systems is that they are not as sensitive as CELISAs.
(V) CELISA and Monoclonal Antibody Conjugate
In the CELISA, the antibody is not captured on the solid matrix by an antibody-specific antibody as in conventional antigen conjugation systems. Instead, an antigen-specific antibody bound to a solid matrix is directly used to capture an antigen-conjugate (see: Voller, A., et al, The Enzyme-Linked Immunosorbent Assay (ELISA), Dynatech Laboratories, Inc. pp. 10-11 (1979) Alexandria, Va.)). The virus antigen is detected by preventing antigen-conjugate binding to the antigen-specific antibody bound to the solid matrix. However, none of the above-described antigen conjugate systems use the CELISA to detect virus-specific antibody. Furthermore, no methods exist for detecting retrovirus core antigen or retrovirus core antigen-specific antibody using a CELISA with or without an antigen conjugate.
A CELISA has been demonstrated for detecting antibody specific for feline infectious peritonitis virus (hereinafter "FIPV") antigens (see: U.S. patent application Ser. No. 716,374, filed Mar. 26, 1985 and Fiscus, S. A., et al, J. Clin. Micro. 22:395-401 1985)). This CELISA uses a virus antigen preparation bound to a solid matrix to capture an enzyme-linked monoclonal antibody specific for a single epitope on a single FIPV antigen. Antibody in the test serum is detected by its competition with the conjugated monoclonal antibody for the virus antigen on the solid matrix. Heretofore, this method has not been demonstrated for the detection of retrovirus core antigen and retrovirus core antigen-specific antibody.